Dynamic modification of nuclear and cytosolic proteins by O-linked N-Acetylglucosamine (O- GlcNAc) is as abundant as protein phosphorylation in all multi-cellular eukaryotes. Two- to five- percent of glucose is metabolized to the end product of the hexosamine biosynthetic pathway (HBP), leading to ~millimolar UDP-GlcNAc in cells. UDP-GlcNAc is the immediate donor for O- GlcNAcylation. O-GlcNAcylation often has an antagonistic, reciprocal ("Yin-Yang") relationship with Ser/Thr phosphorylation. Many laboratories have documented a key role for HBP, and more particularly, O-GlcNAcylation as a mechanism underlying glucose toxicity and insulin resistance, the hallmark of type II diabetes. We propose to continue our studies of the roles of O- GlcNAcylation in insulin signaling and resistance by elucidating the mechanisms by which elevated O-GlcNAc blocks insulin signaling in adipocytes and hepatocytes. We will reveal the site- specific interplay between phosphorylation, and O-GlcNAc's, and elucidate specific roles of each post-translational modification in signaling and transcriptional regulation key to insulin action. Specific Aims: 1) To continue to examine the Structural/Functional Significance of Protein Phosphatase 1 (PP1c) Interactions with O-GlcNAc Transferase (OGT). 2) To continue to elucidate the specific dynamic relationships between O-GlcNAcylation and O-Phosphorylation in the insulin signaling cascade, in order to explain O-GlcNAc's specific roles in hyperglycemic-induced insulin resistance. 3) To continue to examine the roles of O-GlcNAc in the functions/localization/stability and promoter activity of the insulin regulated FOXO1 transcription factor that plays a key role in regulating glucose production by the liver. These studies are revealing an unexpected paradigm for the regulation of signaling pathways by nutrients and by cellular stress. The ability of increased O-GlcNAcylation to block insulin signaling and the modification's sensitivity to stress and nutrients serves to unify current hypotheses of diabetes etiology at the molecular level. These studies will result in novel avenues for therapeutics.